Targeted thrombolytic nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a targeted thrombolytic nano-composite as well as a preparation method and application thereof, belongs to the technical field of biological medicines, and provides the targeted thrombolytic nano-composite which is fluorescent modified ROS (reactive oxygen species) response dendritic mesoporous silicon, ferroferric oxide and a tissue type plasminogen activator are loaded in pores of the dendritic mesoporous silicon, and the dendritic mesoporous silicon is fluorescent modified ROS (reactive oxygen species) response dendritic mesoporous silicon. CREKA polypeptide is connected to the surface of the CREKA polypeptide. The problems that an existing thrombolysis method is poor in targeting property, low in specificity, high in bleeding risk and the like are solved, a targeted thrombolysis nano-composite is fluorescent modified ROS-responded dendritic mesoporous silicon, ferroferric oxide and a tissue-type plasminogen activator are loaded in holes of the dendritic mesoporous silicon, CREKA polypeptide is connected to the surface of the dendritic mesoporous silicon, the drug aggregation and permeability are improved, and the targeting thrombolysis nano-composite has a good application prospect. And a novel chemical and biological integrated thrombolysis direction is provided for a biological targeted intelligent thrombolysis system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a targeted thrombolytic nanocomposite and a preparation method and application thereof. Background Art

[0002] Stroke, also known as cerebral infarction, is commonly known as "stroke" and is also clinically called cerebrovascular accident. It is characterized by high morbidity, mortality, disability and recurrence rates. It is a cerebral blood circulation disorder caused by various inducing factors, such as stenosis, occlusion or rupture of cerebral arteries. It is generally divided into two categories: hemorrhagic stroke and ischemic stroke. Ischemic stroke is a disease in which blood flow to cerebral blood vessels is interrupted due to thrombosis or embolism, causing ischemic and hypoxic damage to brain tissue. Its core mechanism includes the rupture of atherosclerotic plaques to form thrombi (cerebral thrombosis), or the detachment of emboli in parts such as the heart to block distal cerebral blood vessels (cerebral embolism). Exploring and improving emergency treatment strategies for ischemic stroke has always been a hot topic and difficulty in medical research. Currently, intravenous thrombolysis is one of the methods for treating ischemic stroke. However, clinical practice has shown that the recanalization rate of target vessels is low after intravenous injection of anticoagulants (also known as tissue plasminogen activator, tPA), which limits the long-term effect of this treatment.

[0003] The difficulty in recanalizing blood vessels in patients with ischemic stroke is due to several factors. First, during an ischemic stroke, blood flow is interrupted locally due to occlusion of the diseased blood vessels, making it difficult for thrombolytic drugs to reach the vicinity of the clot through the circulation. Second, thrombolytic drugs have a short half-life and are rapidly degraded after intravenous injection, preventing them from forming high concentrations locally at the clot. Therefore, improving the targeting of thrombolytic drugs is crucial for addressing the low recanalization rate. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention proposes a targeted thrombolytic nanocomplex and its preparation method and application, which solves the side effects of existing thrombolytic methods such as poor targeting, low specificity and high risk of bleeding. The targeted thrombolytic nanocomplex is a fluorescently modified ROS-responsive dendritic mesoporous silica, the pores of which are loaded with ferric oxide and tissue plasminogen activator, and the surface is connected with CREKA polypeptide, which improves the drug aggregation and permeability, providing a new thrombolytic direction that integrates chemistry and biology for a biologically targeted intelligent thrombolytic system.

[0005] To achieve the above objectives, the present invention provides a targeted thrombolytic nanocomplex, which is a fluorescently modified ROS-responsive dendritic mesoporous silica, the pores of which are loaded with ferroferric oxide and tissue plasminogen activator, and the surface of which is connected to a CREKA polypeptide.

[0006] Preferably, the fluorescently modified ROS-responsive dendritic mesoporous silica is CY5.5-modified ROS-responsive dendritic mesoporous silica.

[0007] Preferably, the targeted thrombolytic nanocomplex is spherical, with a particle size of 230.403±0.88 nm.

[0008] Preferably, the zeta potential of the targeted thrombolytic nanocomplex is -8.357±3.68 mV.

[0009] The present invention also provides a method for preparing the targeted thrombolytic nanocomposite, comprising the following steps:

[0010] (1) triethanolamine (TEA) and deionized water are mixed and stirred for the first time, and then cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) are added and stirred for the second time to obtain a primary mixed solution;

[0011] (2) adding ethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide to the initial mixed solution obtained in step (1), reacting, and then first centrifuging to obtain a precipitate to obtain a product, and calcining to obtain ROS-responsive dendritic mesoporous silica (MSNP);

[0012] (3) mixing the ROS-responsive dendritic mesoporous silica obtained in step (2) with a fluorescent dye solution, stirring for a third time, and then centrifuging for a second time to obtain a precipitate to obtain fluorescently modified ROS-responsive dendritic mesoporous silica;

[0013] (4) mixing ferroferric oxide with aminopropyltriethoxysilane (APTES), stirring for a fourth time, and then centrifuging for a third time to obtain a precipitate, thereby obtaining amino-modified ferroferric oxide;

[0014] (5) The fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA), CREKA polypeptide (CREKA) obtained in step (3) and the amino-modified ferrosoferric oxide obtained in step (4) are mixed, stirred for a fifth time, and then centrifuged for a fourth time to obtain a precipitate to obtain a targeted thrombolytic nanocomplex (tPA-SPION-MSNP-CREKA).

[0015] Preferably, the mixing ratio of triethanolamine and deionized water in step (1) is 1 g:40 mL; the rotation speed of the first stirring in step (1) is 500 rpm, the temperature of the first stirring is 80° C., and the time of the first stirring is 0.5 h; the ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate in step (1) is 1 g:0.25 g:0.2 mmoL; the rotation speed of the second stirring in step (1) is 800 rpm, the temperature of the second stirring is 30° C., and the time of the second stirring is 1 h.

[0016] Preferably, the ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide in step (2) and triethanolamine used in step (1) is 2g:0.5mL:1g; the reaction temperature in step (2) is 40°C, and the reaction time is 3h; the temperature of the first centrifugation in step (2) is 4°C, the speed of the first centrifugation is 10000rpm, and the time of the first centrifugation is 10min; the calcination temperature in step (2) is 550°C, and the calcination time is 6h.

[0017] Preferably, the ratio of the ROS-responsive dendritic mesoporous silicon and the fluorescent dye solution in step (3) is 5 mg:500 μL, and the concentration of the fluorescent dye solution in step (3) is 0.02 mmol / mL; the speed of the third stirring in step (3) is 600 rpm, the temperature of the third stirring is 25°C, and the time of the third stirring is 2 h; the temperature of the second centrifugation in step (3) is 4°C, the speed of the second centrifugation is 12000 rpm, and the time of the second centrifugation is 10 min.

[0018] Preferably, the mixing ratio of ferroferric oxide and aminopropyltriethoxysilane in step (4) is 10 mg:100 μL; the rotation speed of the fourth stirring in step (4) is 800 rpm, the temperature of the fourth stirring is 70°C, and the time of the fourth stirring is 3 h; the temperature of the third centrifugation in step (4) is 4°C, the rotation speed of the third centrifugation is 12000 rpm, and the time of the third centrifugation is 10 min; the mixing ratio of the fluorescent modified ROS-responsive dendritic mesoporous silica, tissue-type plasminogen activator, CREKA polypeptide obtained in step (3) and the amino-modified ferroferric oxide obtained in step (4) in step (5) is 5 mg:100 μg:0.01 mmoL:5 mg; the rotation speed of the fifth stirring in step (5) is 600 rpm, the temperature of the fifth stirring is 4°C, and the time of the fifth stirring is 2 h; the temperature of the fourth centrifugation in step (5) is 4°C, the rotation speed of the fourth centrifugation is 12000 rpm, and the time of the fourth centrifugation is 10 min.

[0019] The present invention also provides the use of the targeted thrombolytic nanocomplex in preparing a drug for treating ischemic stroke.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention provides a targeted thrombolytic nanocomposite, which modifies ROS-responsive dendritic mesoporous silica with a targeting peptide CREKA polypeptide (Cys-Arg-Glu-Lys-Ala) that can specifically recognize thrombus fibrin. By leveraging the high specific surface area and strong loading capacity of mesoporous silica, the thrombolytic drug TPA and superparamagnetic ferroferric oxide are simultaneously loaded in its mesopores. This design significantly increases the local TPA concentration in the thrombus, prolongs the plasma half-life of the thrombolytic drug, increases the safe dosage of the thrombolytic drug, and reduces the risk of post-thrombolytic bleeding. At the same time, due to the magnetic effect of superparamagnetic ferroferric oxide, the drug's permeability to the thrombus is improved, thereby improving the prognosis of ischemic stroke.

[0022] The targeted thrombolytic nanocomposite of the present invention has a strong loading capacity. The mesoporous silica can accommodate a large number of drug molecules by utilizing the high specific surface area and porous structure of the mesoporous silica. It has the function of rapidly targeting thrombi. The ROS-responsive dendritic mesoporous silica is modified with CREKA. The specific binding of CREKA to fibrin in the thrombus is utilized to increase the aggregation rate of the thrombolytic drug around the thrombus. The peripheral degradation of the thrombolytic drug is reduced. The narrow mesopores of the ROS-responsive dendritic mesoporous silica are utilized to limit the structural unfolding of the protein (TPA), thereby reducing the peripheral degradation of TPA. It has a controlled release characteristic. When the drug reaches the vicinity of the thrombus, the drug molecules are slowly released by stimulating the alternating magnetic field, showing a low initial burst release and a continuous sustained release characteristic, which makes the drug release more stable and lasting. It has a synergistic thrombolytic effect. The ROS-responsive dendritic mesoporous silica loaded with TPA and the surface modified CY5.5 of ferroferric oxide combined with the alternating magnetic field stimulation can enhance the release of TPA. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Schematic diagram of the synthesis process of the targeted thrombolytic nanocomplex tPA-SPION-MSNP-CREKA of the present invention;

[0025] Figure 2 The particle size distribution and potential diagrams of the MSNPs prepared in Example 1, the tPA-SPION-MSNPs prepared in Comparative Example 1, and the tPA-SPION-MSNP-CREKA prepared in Example 1, wherein A is the particle size distribution diagram and B is the potential diagram;

[0026] Figure 3Scanning electron micrographs and transmission electron micrographs of the MSNPs prepared in Example 1, the tPA-SPION-MSNPs prepared in Comparative Example 1, and the tPA-SPION-MSNP-CREKA prepared in Example 1, wherein A is a transmission electron micrograph of the MSNPs prepared in Example 1, B is a transmission electron micrograph of the tPA-SPION-MSNPs prepared in Comparative Example 1, C is a transmission electron micrograph of the tPA-SPION-MSNP-CREKA prepared in Example 1, D is a scanning electron micrograph of the MSNPs prepared in Example 1, E is a scanning electron micrograph of the tPA-SPION-MSNPs prepared in Comparative Example 1, and F is a scanning electron micrograph of the tPA-SPION-MSNP-CREKA prepared in Example 1. The scale is 200 nm.

[0027] Figure 4 The fibrinolysis plate test results of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1, wherein A is a physical image of the fibrinolysis plate test, B is a quantitative statistical graph of the diameter of the transparent circle, and "*" in the figure represents a significant difference analysis, C is an image of the binding of fluorescent-labeled nanoparticles on the fibrin gel plate, and D is a statistical graph of the average fluorescence intensity of the fluorescence image, and "*" in the figure represents a significant difference analysis;

[0028] Figure 5 These are the in vitro thrombolytic ability test results of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1, wherein A is a comparison of thrombi in each group after 6 hours of treatment and thrombi before thrombolysis, B is the thrombolysis percentage in each group after 6 hours of treatment, and "*" and "**" in the figure represent significant difference analysis, C is an observation graph of fibrin release in the supernatant during 6 hours of thrombus treatment in each group, and D is a statistical graph of fibrin content released in the supernatant during 6 hours of thrombus treatment in each group, and "*", "**" and p=0.7 in the figure represent significant difference analysis;

[0029] Figure 6 The results of in vivo thrombolytic ability test of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown, wherein A is the cerebral blood perfusion image (LSCI) of mice in each group, and B is the difference in cerebral infarction volume shown by TTC staining of the brains of mice in each group;

[0030] Figure 7These are the results of in vivo thrombolytic ability analysis of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1, where A is the trend of changes in cerebral blood perfusion (CBF) in different treatment groups at different time points after surgery, and B is a comparison of the percentage of cerebral infarction volume in mice in different treatment groups. The "*" in the figure represents the significance analysis of the difference. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The sources of materials used in the present invention are as follows: triethanolamine was purchased from Sinopharm Chemical Reagent Co., Ltd.; cetyltrimethylammonium bromide was purchased from Aladdin Reagent Co., Ltd.; sodium salicylate was purchased from Sinopharm Chemical Reagent Co., Ltd.; ethyl silicate was purchased from Aladdin Reagent Co., Ltd.; bis-[3-(triethoxysilyl)propyl]-disulfide was purchased from Xi'an Ruixi; CY5.5 was purchased from Beyotime Biotechnology Co., Ltd.; ferrous oxide was purchased from Xi'an Ruixi; aminopropyltriethoxysilane was purchased from Aladdin Reagent Co., Ltd.; tissue plasminogen activator was purchased from Boehringer Ingelheim; and CREKA polypeptide was purchased from Xi'an Ruixi.

[0037] Example 1

[0038] (1) Triethanolamine (TEA) and deionized water were mixed at a ratio of 1 g:40 mL and stirred at 500 rpm and 80°C for 0.5 h. Cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) were then added and stirred at 800 rpm and 30°C for 1 h to obtain a primary mixed solution. The ratio of triethanolamine, cetyltrimethylammonium bromide, and sodium salicylate was 1 g:0.25 g:0.2 mmol / L.

[0039] (2) Ethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide were added to the initial mixed solution and reacted at 40°C for 3 h. The mixture was then centrifuged at 4°C and 10,000 rpm for 10 min. The precipitate was washed three times with deionized water and ethanol alternately to remove the residual reactants. The product was then calcined at 550°C for 6 h to remove the structure-directing agent, thereby obtaining ROS-responsive dendritic mesoporous silica (MSNP). The ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine was 2 g:0.5 mL:1 g.

[0040] (3) The ROS-responsive dendritic mesoporous silica was mixed with CY5.5 solution at a ratio of 5 mg:500 μL, the concentration of the CY5.5 solution was 0.02 mmol / mL, and the mixture was stirred for a third time at 25°C and 600 rpm for 2 h, and then centrifuged for a second time at 4°C and 12000 rpm for 10 min to remove the unloaded CY5.5, and the precipitate was obtained to obtain the fluorescently modified ROS-responsive dendritic mesoporous silica.

[0041] (4) Ferroferric oxide and aminopropyltriethoxysilane (APTES) were mixed in a ratio of 10 mg:100 μL, stirred at 70°C and 800 rpm for 3 h, and then centrifuged at 4°C and 12,000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain amino-modified ferroferric oxide.

[0042] (5) Fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA), CREKA polypeptide (CREKA) and amino-modified ferrosoferric oxide were mixed in a ratio of 5 mg:100 μg:0.01 mmoL:5 mg, stirred for 2 h at 4°C and 600 rpm, and then centrifuged for 10 min at 4°C and 12,000 rpm. The precipitate was collected and washed with deionized water to obtain a targeted thrombolytic nanocomplex (tPA-SPION-MSNP-CREKA).

[0043] like Figure 1 Shown is a schematic diagram of the synthesis process of the targeted thrombolytic nanocomplex (tPA-SPION-MSNP-CREKA).

[0044] Comparative Example 1

[0045] (1) Triethanolamine (TEA) and deionized water were mixed at a ratio of 1 g:40 mL and stirred at 500 rpm and 80°C for 0.5 h. Cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) were then added and stirred at 800 rpm and 30°C for 1 h to obtain a primary mixed solution. The ratio of triethanolamine, cetyltrimethylammonium bromide, and sodium salicylate was 1 g:0.25 g:0.2 mmol / L.

[0046] (2) Ethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide were added to the initial mixed solution and reacted at 40°C for 3 h. The mixture was then centrifuged at 4°C and 10,000 rpm for 10 min. The precipitate was washed three times with deionized water and ethanol alternately to remove the residual reactants. The product was then calcined at 550°C for 6 h to remove the structure-directing agent, thereby obtaining ROS-responsive dendritic mesoporous silica (MSNP). The ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine was 2 g:0.5 mL:1 g.

[0047] (3) The ROS-responsive dendritic mesoporous silica was mixed with CY5.5 solution at a ratio of 5 mg:500 μL, the concentration of the CY5.5 solution was 0.02 mmol / mL, and the mixture was stirred for a third time at 25°C and 600 rpm for 2 h, and then centrifuged for a second time at 4°C and 12000 rpm for 10 min to remove the unloaded CY5.5, and the precipitate was obtained to obtain the fluorescently modified ROS-responsive dendritic mesoporous silica.

[0048] (4) Ferroferric oxide and aminopropyltriethoxysilane (APTES) were mixed in a ratio of 10 mg:100 μL, stirred at 70°C and 800 rpm for 3 h, and then centrifuged at 4°C and 12,000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain amino-modified ferroferric oxide.

[0049] (5) Fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA) and amino-modified ferrosoferric oxide were mixed in a ratio of 5 mg:100 μg:5 mg, stirred for 2 h at 4°C and 600 rpm, and then centrifuged for 10 min at 4°C and 12,000 rpm. The precipitate was collected and washed with deionized water to obtain a targeted thrombolytic nanocomplex (tPA-SPION-MSNP).

[0050] Experimental Example 1

[0051] The particle size and potential of MSNPs, tPA-SPION-MSNPs, and tPA-SPION-MSNP-CREKA were measured by a nanoparticle size potentiostat (NanoBrook 90plus PALS). The MSNPs, tPA-SPION-MSNPs, and tPA-SPION-MSNP-CREKA were characterized by TEM and SEM.

[0052] like Figure 2 As shown in Figure A, the particle sizes of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA are 232.433±2.35nm, 232.023±8.55nm, and 230.403±0.88nm, respectively, indicating that the introduction of polypeptide chains did not significantly change the particle size of the nanoparticles. Figure 2 As shown in Figure 2B, the zeta potentials of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA were -10.77±0.21 mV, -13.023±0.71 mV, and -8.357±3.68 mV, respectively.

[0053] like Figure 3 As shown, Figure 3 Middle A, Figure 3 Middle B and Figure 3 C in the middle are TEM images of MSNP, tPA-SPION-MSNP and tPA-SPION-MSNP-CREKA, respectively. Figure 3 Middle A shows the transmission electron microscope image of blank mesoporous silica nanoparticles (MSNPs), which show a regular spherical morphology with a typical dendritic radial pore structure on the surface, which is evenly distributed. The particle size is about 200 nm. Figure 3 Figure B shows the MSNP loaded with superparamagnetic iron oxide nanoparticles (SPION) and tissue plasminogen activator (tPA) (i.e., tPA-SPION-MSNP). It can be observed that there are black high electron density areas on the surface of some particles, which are the loading signals of SPION. Figure 3Center C shows the nanoparticle tPA-SPION-MSNP-CREKA, which has been further modified with the CREKA peptide. Compared to tPA-SPION-MSNP, more black clusters of high-density regions are visible on its surface, indicating that the CREKA peptide has been successfully modified onto the carrier surface. Furthermore, the pores of some nanoparticles are blurred, suggesting that the CREKA modification has a certain impact on the local structure, possibly enhancing the compactness and stability of the carrier structure. Figure 3 Middle D, Figure 3 Zhong E and Figure 3 Figures F in the middle are SEM images of MSNP, tPA-SPION-MSNP and tPA-SPION-MSNP-CREKA, respectively. From the SEM images, it can be directly observed that the surfaces of the MSNP prepared in Example 1, the tPA-SPION-MSNP prepared in Comparative Example 1 and the tPA-SPION-MSNP-CREKA prepared in Example 1 contain a large number of pores, and the pores on the surface of tPA-SPION-MSNP-CREKA have disappeared, indicating that the modification of the CREKA polypeptide improves the stability of the nano-ionic structure.

[0054] Experimental Example 2

[0055] Biological targeting and in vitro fibrinolytic capacity testing:

[0056] To verify whether the targeted thrombolytic nanocomplex has a biological targeting effect on thrombi, a fibrin gel containing fibrinogen and thrombin was first prepared at 37°C. Equidistant small holes were punched in the completely coagulated fibrin gel using a 3mm needle, leaving 6 test holes per dish. Subsequently, TPA, an equal amount of tPA-SPION-MSNP-CREKA prepared in Example 1, or an equal amount of tPA-SPION-MSNP prepared in Comparative Example 1 were added dropwise and incubated at 37°C for 30 minutes to ensure sufficient binding to fibrin. After incubation, the mixture was gently rinsed three times with 1mL of PBS to remove nonspecifically adsorbed particles. The fibrinolytic activity of different drugs was compared by measuring the diameter of the fibrinolytic zone. Three random fields of view were collected on each plate under the same light source and exposure parameters using a fluorescence microscope (λ_ex = 488nm). The total fluorescence integrated density was quantified using ImageJ. The fibrinolytic activity of different drugs was compared by measuring the diameter of the fibrinolytic zone.

[0057] like Figure 4 Middle A, Figure 4 Middle B, Figure 4 Middle C and Figure 4As shown in D, the transparent aperture of tPA-SPION-MSNP-CREKA is twice that of tPA-SPION-MSNP, and the average fluorescence intensity of tPA-SPION-MSNP-CREKA on the fiber plate is about 1.5 times that of tPA-SPION-MSNP, and the difference is statistically significant (P < 0.05, n = 3 fields of view).

[0058] Transparent aperture diameter Figure 4 China A and Figure 4 As shown in Figure B, the diameters of the transparent zones formed by tPA and tPA-SPION-MSNP are similar (≈0.6 cm), indicating that the thrombolytic activity of tPA is not significantly reduced after encapsulation. The diameter of the transparent zone of tPA-SPION-MSNP-CREKA is significantly increased to approximately 2 times that of tPA-SPION-MSNP (P < 0.05, n = 3), indicating that under the same enzyme activity conditions, CREKA modification significantly enhances the local fibrinolytic ability of tPA. Figure 4 Middle C and Figure 4 As shown in Figure D, the average fluorescence intensity of tPA-SPION-MSNP-CREKA on the fibrin mesh was approximately 1.5 times that of tPA-SPION-MSNP (P < 0.05, n = 3 fields of view), indicating that the CREKA peptide significantly enhanced the specific adsorption of the nanocarrier to the fibrin mesh. Combined clearing zone and fluorescence quantitative experiments demonstrated that CREKA efficiently enriched tPA-SPION-MSNP on the surface of the fibrin mesh, increasing local enzyme concentration and amplifying the fibrinolytic effect at the same dosage. These results demonstrate that the CREKA peptide significantly enhances the specific binding ability of TPA to the fibrin mesh, validating its superior biological targeting.

[0059] Experimental Example 3

[0060] In vitro thrombolytic ability test:

[0061] 120 mg of fresh mouse thrombus prepared in advance was placed in EP tubes containing 1 mL of normal saline, TPA (0.2 mg / mL), tPA-SPION-MSNP prepared in Comparative Example 1 (free TPA concentration 0.2 mg / mL), and tPA-SPION-MSNP-CREKA prepared in Example 1 (free TPA concentration 0.2 mg / mL) and incubated in a 37°C water bath. The thrombus mass was measured after 6 hours of treatment and compared with the thrombus mass before thrombolysis to calculate the thrombolysis percentage; the absorbance (OD) of the supernatant at 410 nm at different time points was measured to measure the amount of fibrin released from the thrombus, and then the thrombolytic effect was analyzed.

[0062] like Figure 5 China A and Figure 5As shown in B, the thrombus mass after 6 hours of treatment was compared with the thrombus mass before thrombolysis to calculate the thrombolysis percentage. The thrombus volume became smaller and thinner with time, and the supernatant of the solution changed from colorless to deep red. Figure 5 Middle C and Figure 5 As shown in Figure D, the study found that starting at 4 hours of treatment, the supernatant of the tPA-SPION-MSNP-CREKA group released significantly more fibrin than the tPA-SPION-MSNP group. Furthermore, after 6 hours of treatment, the thrombus had become significantly thinner and softer, and the thrombus dissolution percentage in the tPA-SPION-MSNP-CREKA group was significantly higher than that in the tPA-SPION-MSNP group. This result suggests that tPA-SPION-MSNP-CREKA treatment can enhance the thrombolytic effect.

[0063] Experimental Example 4

[0064] In vivo thrombolytic ability test:

[0065] In order to study the thrombolytic ability of tPA-SPION-MSNP-CREKA prepared in Example 1 in vivo, it was evaluated in a mouse model of MCAO. The model mice were randomly divided into 4 groups and treated with equal amounts of normal saline, TPA, tPA-SPION-MSNP prepared in Comparative Example 1, and tPA-SPION-MSNP-CREKA prepared in Example 1. Taking tPA-SPION-MSNP-CREKA as an example, 10 minutes after the operation, after the laser speckle blood flow imaging system confirmed the success, tPA-SPION-MSNP-CREKA (1 mg tPA kg -1 The dosage was calculated based on tPA equivalents, with the tPA content in the formulation being: 5 mg of carrier ≈ 100 μg of tPA. A magnet was used to guide the tPA-SPION-MSNP-CREKA to the M1 segment of the middle cerebral artery, the site of the thrombus. Regional cerebral blood flow was monitored before surgery and at 0, 3, and 6 hours after surgery, and changes in cerebral blood flow were compared among the four groups.

[0066] The results are as follows Figure 6 Middle A, Figure 6 Middle B, Figure 7 China A and Figure 7 As shown in Figure B, the study found that tPA-SPION-MSNP-CREKA significantly restored cerebral blood flow 6 hours after surgery; mNSS was used to evaluate neurological deficits 6 hours after surgery, and TTC was used to compare cerebral infarction volumes. The infarction volume in the tPA-SPION-MSNP-CREKA group was smaller than that in the tPA-SPION-MSNP group. The above studies show that the synergistic effect of CREKA improves neurological damage at the in vivo level.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A targeted thrombolytic nanocomposite, characterized in that: The targeted thrombolytic nanocomposite is a fluorescently modified ROS-responsive dendritic mesoporous silica, pores of which are loaded with ferroferric oxide and tissue-type plasminogen activator, and the surface of the complex is connected with a CREKA polypeptide.

2. The targeted thrombolytic nanocomposite according to claim 1, characterized in that: The fluorescent modified ROS-responsive dendritic mesoporous silica is CY5.5-modified ROS-responsive dendritic mesoporous silica.

3. The targeted thrombolytic nanocomposite according to claim 1, characterized in that: The targeted thrombolytic nanocomplex is spherical and has a particle size of 230.403±0.88 nm.

4. The targeted thrombolytic nanocomposite according to claim 1, characterized in that: The zeta potential of the targeted thrombolytic nanocomplex is -8.357±3.68 mV.

5. The method for preparing the targeted thrombolytic nanocomposite according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) triethanolamine and deionized water are mixed and stirred for the first time, and then cetyltrimethylammonium bromide and sodium salicylate are added and stirred for the second time to obtain a primary mixed solution; (2) adding ethyl silicate and bis-[3-(triethoxysilyl)propyl]-disulfide to the initial mixed solution obtained in step (1), reacting, and then first centrifuging to obtain a precipitate to obtain a product, and calcining to obtain ROS-responsive dendritic mesoporous silica; (3) mixing the ROS-responsive dendritic mesoporous silica obtained in step (2) with a fluorescent dye solution, stirring for a third time, and then centrifuging for a second time to obtain a precipitate to obtain fluorescently modified ROS-responsive dendritic mesoporous silica; (4) mixing ferrosoferric oxide with aminopropyltriethoxysilane, stirring for a fourth time, and then centrifuging for a third time to obtain a precipitate, thereby obtaining amino-modified ferrosoferric oxide; (5) The fluorescently modified ROS-responsive dendritic mesoporous silica, tissue-type plasminogen activator, CREKA polypeptide obtained in step (3) and the amino-modified ferrosoferric oxide obtained in step (4) are mixed, stirred for a fifth time, and then centrifuged for a fourth time to obtain a precipitate, thereby obtaining a targeted thrombolytic nanocomplex.

6. The preparation method according to claim 5, characterized in that: The mixing ratio of triethanolamine and deionized water in step (1) is 1 g:40 mL; the rotation speed of the first stirring in step (1) is 500 rpm, the temperature of the first stirring is 80° C., and the time of the first stirring is 0.5 h; the ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate in step (1) is 1 g:0.25 g:0.2 mmoL; the rotation speed of the second stirring in step (1) is 800 rpm, the temperature of the second stirring is 30° C., and the time of the second stirring is 1 h.

7. The preparation method according to claim 5, characterized in that: The ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide and triethanolamine used in step (1) in step (2) is 2g:0.5mL:1g; the reaction temperature in step (2) is 40°C, and the reaction time is 3h; the temperature of the first centrifugation in step (2) is 4°C, the speed of the first centrifugation is 10000rpm, and the time of the first centrifugation is 10min; the calcination temperature in step (2) is 550°C, and the calcination time is 6h.

8. The preparation method according to claim 5, characterized in that: The ratio of the ROS-responsive dendritic mesoporous silicon and the fluorescent dye solution in step (3) is 5 mg:500 μL, and the concentration of the fluorescent dye solution in step (3) is 0.02 mmol / mL; the speed of the third stirring in step (3) is 600 rpm, the temperature of the third stirring is 25°C, and the time of the third stirring is 2 h; the temperature of the second centrifugation in step (3) is 4°C, the speed of the second centrifugation is 12000 rpm, and the time of the second centrifugation is 10 min.

9. The preparation method according to claim 5, characterized in that: The mixing ratio of ferroferric oxide and aminopropyltriethoxysilane in step (4) is 10 mg:100 μL; the rotation speed of the fourth stirring in step (4) is 800 rpm, the temperature of the fourth stirring is 70°C, and the time of the fourth stirring is 3 h; the temperature of the third centrifugation in step (4) is 4°C, the rotation speed of the third centrifugation is 12000 rpm, and the time of the third centrifugation is 10 min; the mixing ratio of the fluorescent modified ROS-responsive dendritic mesoporous silica, tissue-type plasminogen activator, CREKA polypeptide obtained in step (3) and the amino-modified ferroferric oxide obtained in step (4) in step (5) is 5 mg:100 μg:0.01 mmoL:5 mg; the rotation speed of the fifth stirring in step (5) is 600 rpm, the temperature of the fifth stirring is 4°C, and the time of the fifth stirring is 2 h; the temperature of the fourth centrifugation in step (5) is 4°C, the rotation speed of the fourth centrifugation is 12000 rpm, and the time of the fourth centrifugation is 10 min.

10. Use of the targeted thrombolytic nanocomplex according to any one of claims 1 to 4 in the preparation of a drug for treating ischemic stroke.

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